Transgenic mice over-expressing receptor for advanced glycation endproduct (RAGE) in brain and uses thereof

ABSTRACT

The present invention provides for a transgenic non-human animal whose cells contain a DNA sequence comprising: (a) a nerve tissue specific promoter; and (b) a DNA sequence which encodes a receptor for advanced glycation endproducts (RAGE), wherein the promoter and the DNA sequence which encodes the receptor for advanced glycation endproducts (RAGE) are operatively linked to each other and integrated in the genome of the non-human animal, and wherein said non-human animal exhibits a reduced amount of cerebral tissue infarcted following a transient middle cerebral artery occlusion compared to an identical non-human animal lacking said DNA sequence.

BACKGROUND OF THE INVENTION

Throughout this application, various publications are referenced bynumber. Full citations for these publications may be found listed at theend of the specification immediately preceding the claims. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art as known to those skilled therein as ofthe date of the invention described and claimed herein.

The pain of Alzheimer's disease results directly from the memory lossand cognitive deficits suffered by the patient. These eventually resultin the patient's loss of identity, autonomy, and freedom. As a steptoward curing this disease, alleviating its symptoms, or retarding itsprogression, it would be desirable to develop a transgenic animal modelexhibiting the main debilitating phenotype of Alzheimer's disease, thatis, memory loss, expressed concomitantly with the neuropathologicalcorrelates of Alzheimer's disease, for example, beta-amyloidaccumulation, increased glial reactivity, and hippocampal cell loss.

It is estimated that over 5% of the U.S. population over 65 and over 15%of the U.S. population over 85 are beset with some form of Alzheimer'sdisease (Cross, A. J., Eur J Pharmacol (1982) 82:77-80; Terry, R. D., etal., Ann Neurol (1983) 14:497506). It is believed that the principalcause for confinement of the elderly in long term care facilities is dueto this disease, and approximately 65% of those dying in skilled nursingfacilities suffer from it.

Certain facts about the biochemical and metabolic phenomena associatedwith the presence of Alzheimer's disease are known. Two morphologicaland histopathological changes noted in Alzheimer's disease brains areneurofibrillary tangles (NFT) and amyloid deposits. Intraneuronalneurofibrillary tangles are present in other degenerative diseases aswell, but the presence of amyloid deposits both in the interneuronalspaces (neuritic plaques) and in the surrounding microvasculature(vascular plaques) seems to be characteristic of Alzheimer's. Of these,the neuritic plaques seem to be the most prevalent (Price, D. L., etal., Drug Development Research (1985) 5:59-68). Plaques are also seen inthe brains of aged Down's Syndrome patients who develop Alzheimer'sdisease.

SUMMARY OF THE INVENTION

The present invention provides for a transgenic non-human animal whosecells contain a DNA sequence comprising: (a) a nerve tissue specificpromoter; and (b) a DNA sequence which encodes a receptor for advancedglycation endproducts (RAGE), wherein the promoter and the DNA sequencewhich encodes the receptor for advanced glycation endproducts (RAGE) areoperatively linked to each other and integrated in the genome of thenon-human animal, and wherein said non-human animal exhibits a reducedamount of cerebral tissue infarcted following a transient middlecerebral artery occlusion compared to an identical non-human animallacking said DNA sequence.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1B. Schematic depiction of strategy for making Tg PD-RAGE mice.

FIG. 2. Southern analysis of three founders for Tg PD-RAGE mice: lanes1, 3, 6 show mice positive for the transgene and lanes 2, 4-5 arenontransgenic littermates.

FIG. 3. Identification of Tg PD-RAGE mice (+) and nontransgeniclittermate controls (−) by PCR.

FIGS. 4A-4B. RAGE expression in Tg PD-RAGE mice (+) compared withnontransgenic littermate controls (−). A (Northern) and B (Western)analysis of homogenates of cerebral cortex. Equal amounts of RNA (noteapproximately equal intensity of 28S ribosomal RNA band on the ethidiumbromide stained gel) and protein were loaded in each lane.

FIG. 5. RAGE expression in brain subregions of Tg PD-RAGE mice comparedwith nontransgenic littermate controls (nonTg). Immunoblotting wasperformed protein extracts of brain homogenates derived from theindicated brain subregion.

FIGS. 6A-6B. Immunohistochemical identification of RAGE in cerebralcortex from a Tg PD-RAGE mouse (FIG. 6A) and a nontransgenic littermatecontrol (FIG. 6B).

FIG. 7. Transient middle cerebral artery occlusion model of stroke inmice: comparison of infarct volume in Tg PD-RAGE and nontransgeniclittermate controls (nonTg). *P<0.05.

FIG. 8. Identification of double transgenic mice overexpressing RAGE andmutant human APP by PCR.

FIGS. 9A, 9B1, 9B2, 9B3, 9C. Increased expression of M-CSF in cerebralcortex from double Tg mice overexpressing RAGE and mutant human APP(hAPP). FIG. 9A, Northern analysis for M-CSF transcripts. FIGS. 9B1-9B3,immunostaining for M-CSF. FIG. 9C, Quantitation of immunocytochemicalresults.

FIGS. 10A, 10B1, 10B2, 10B3, 10C. Increased expression of Interleukin(IL)-6 in cerebral cortex from double Tg mice overexpressing RAGE andmutant human APP (hAPP). FIG. 10A, Northern analysis for IL-6transcripts. FIGS. 10B1-10B3, immunostaining for IL-6. FIG. 10C,Quantitation of immunocytochemical results.

FIG. 11. EMSA for NF-kB on nuclear extracts from cerebral cortex of miceoverexpressing RAGE (2,3), mutant human APP (hAPP; 4,5), both transgenes(6-9), and nontransgenic littermate control (1).

FIG. 12. Semiquantitative analysis of synaptophysin immunoreactivity inhippocampus of Tg PD-RAGE/hAPP, Tg PD-RAGE, Tg hAPP, and nontransgeniclittermate control mice at 4 months of age.

FIG. 13. Semiquantitative analysis of MAP-2 immunoreactivity inhippocampus of Tg PD-RAGE/hAPP, Tg PD-RAGE, Tg hAPP, and nontransgeniclittermate control mice at 4 months of age.

FIGS. 14A1, 14A2, 14A3, 14A4, and 14B. Increased expression of activatedcaspase-3 in cerebral cortex from Tg PD-RAGE/hAPP mice. FIGS. 14A1-4,immunostaining for activated caspase-3. FIG. 14B, quantitation ofimmunocytochemical results from multiple fields of all mice in each ofthe experimental groups. Scale bar, 10 μm.

FIGS. 15A1, 15A2, 15A3, 15A4, and 15B. Immunostaining (FIGS. 15A1-4)with antibody to phosphorylated tau (AT8) in cerebral cortex of theindicated transgenic mice. FIG. 15B demonstrates image analysis ofmultiple microscopic fields from all of the mice in each of theexperimental groups. Scale bar, 10 μm.

FIG. 16. Immunoblotting of E16 cortical neuron cultures with anti-humanRAGE IgG. (+) indicates neurons obtained from Tg PD-RAGE mice and (−)indicates neurons are from nontransgenic littermate controls.

FIGS. 17A-17B. NF-kB activation in primary cortical neuron cultures fromTg PD-RAGE and nontransgenic littermates exposed for 5 hrs to preformedAβ(1-40) fibrils (500 nM) alone (FIG. 17A (left panel)) or in thepresence of anti-RAGE IgG or nonimmune (NI) IgG (FIG. 17B (rightpanel)). Gel shift analysis was performed with ³²P-labelled NF-kB probe.

FIG. 18. Cortical neuron cultures (as in FIGS. 16-17) were exposed topreformed Aβ(1-40) fibrils (2 μM) for 30 or 40 hours, and caspase-3activity was monitored. Neurons were derived from Tg PD-

FIGS. 19A-19B. Volume of infarcted cerebral tissue was reduced in RAGEoverexpressing transgenic mice compared with the control mice. Thevolume was reduced about 50% (p<0.05) in the transgenic mice comparedwith normal mice. FIG. 19A shows results of studies in all mice; FIG.19B shows triphenyl tetrazolium chloride staining of selected cerebralsections.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides for a transgenic non-human animal whosecells contain a DNA sequence comprising: (a) a nerve tissue specificpromoter; and (b) a DNA sequence which encodes a receptor for advancedglycation endproducts (RAGE), wherein the promoter and the DNA sequencewhich encodes the receptor for advanced glycation endproducts (RAGE) areoperatively linked to each other and integrated in the genome of thenon-human animal, and wherein said non-human animal exhibits a reducedamount of cerebral tissue infarcted following a transient middlecerebral artery occlusion compared to an identical non-human animallacking said DNA sequence.

In one embodiment of the invention, the promoter is platelet derivedgrowth factor (PDGF)-B-chain promoter. In another embodiment of theinvention, the DNA sequence which encodes amyloid-beta peptide alcoholdehydrogenase is a human DNA sequence. In another embodiment, thereduction of infarcted cerebral tissue is about a 50% reduction. Inanother embodiment, the transgenic non-human animal is a mouse, a rat, asheep, a dog, a primate, or a reptile. In another embodiment of theinvention, the non-human animal is a mammal.

This invention also provides for a method for evaluating in a non-humantransgenic animal the potential therapeutic effect of an agent fortreating Alzheimer's disease in a human, which comprises: (a)administering an agent to a transgenic non-human animal whose cellscomprise a nerve tissue specific promoter operatively linked to a DNAsequence which encodes receptor for advanced glycation endproducts(RAGE); and (b) determining the therapeutic effect of the agent on thetransgenic non-human animal by monitoring basal synaptic transmission orsynaptic plasticity, wherein an increase in basal synaptic transmissionor synaptic plasticity indicates that the agent would have a potentialtherapeutic effect on Alzheimer's disease in a human.

The invention also provides for a method for identifying whether anagent or a compound is an inhibitor of receptor for advanced glycationendproduct (RAGE) in vivo, which comprises (a) obtaining a non-humantransgenic animal whose cells overexpress RAGE in neurons; (b)administering an agent or compound to the transgenic non-human animal;(c) determining whether the transgenic non-human animal from step (b)exhibits a change in neuronal function from an identical transgenicnon-human animal which was not administered the agent or compound;wherein a determination of change in neuronal function indicates thatthe agent or compound is an inhibitor of RAGE in vivo.

In one embodiment of the invention, the promoter of both element (a) and(b) is platelet derived growth factor (PDGF)-B-chain promoter.

In another embodiment of the invention, the non-human animal is a mouse,a rat, a sheep, a dog, a primate, or a reptile. In another embodiment,the animal is a mammal.

The phenotype observed in the transgenic RAGE overexpressing micedescribed herein was not obvious prior to the creation of such mice. Thetransgenic mice described herein only overexpress RAGE in neurons,whereas in the normal animal, RAGE is also expressed in the microglia athigh levels (the microglia are considered important cells in thepathogenesis of Alzheimer's disease). Therefore, prior to creating andstudying the actual transgenic, one could have imagined thatoverexpression of RAGE in neurons alone would not have had a significanteffect on the resulting transgenic animal. However, as describedhereinbelow, there is evidence that RAGE overexpressing mice exhibit areduced neurologic deficit score and that RAGE overexpressing mice havea reduced volume of infarcted cerebral tissue when subjected to thetransient middle cerebral artery occlusion procedure (described below).

Nucleotide and Amino Acid sequences of RAGE

The nucleotide and protein (amino acid) sequences for RAGE (both humanand murine and bovine) are known. The following references which recitethese sequences are incorported by reference:

Schmidt et al, Biol. Chem., 267:14987-97, 1992

Neeper et al, J. Biol. Chem., 267:14998-15004, 1992

RAGE sequences (DNA sequence and translation) from bovine, murine andhomo sapien are listed hereinbelow. These sequences are available fromGenBank as are other sequences of RAGE from other species:

LOCUS BOVRAGE 1426 by mRNA MAM 09-DEC-1993 DEFINITION Cow receptor foradvanced glycosylation end products (RAGE) mRNA, complete cds.

ACCESSION M91212VERSION M91212.1 GI:163650

KEYWORDS RAGE; cell surface receptor.

SOURCE Bos taurus cDNA to mRNA. ORGANISM Bos taurus Eukaryota; Metazoa;Chordata; Craniata, Vertebrata; Euteleostomi; Mammalia; Eutheria;Cetartiodactyla; Ruminantia; Pecora; Bovoidea; Bovidae; Bovinae; Bos.

REFERENCE 1 (bases 1 to 1426) AUTHORS Neeper, M., Schmidt, A. M., Brett,J., Yan, S. D., Wang, F., Pan, Y. C., Elliston, K., Stern, D. and Shaw,A. TITLE Cloning and expression of a cell surface receptor for advancedglycosylation end products of proteins

JOURNAL J. Biol. Chem. 267, 14998-15004 (1992)

MEDLINE 92340547 REFERENCE 2 (bases 1 to 1426) AUTHORS Shaw, A. TITLEDirect Submission JOURNAL Submitted (15-APR-1992) A. Shaw, Department ofCellular and Molecular Biology, Merck Sharp and Dohme ResearchLaboratories, West Point, Pa. 19486

USAFEATURES Location/Qualifiers source 1..1426 /organism=“Bos taurus”/db_xref=“taxon:9913” /tissue_type=“lung” CDS 10..1260/standard_name=“RAGE” /codon_start=1 /product=“receptor for advancedglycosylation end products” /protein_id=“AAA03575.1”/db_xref=“GI:163651”/translation=”

(SEQ ID NO: 1) MAAGAVVGAWMLVLSLGGTVTGDQNITARIGKPLVLNCKGAPKKPPQQLEWKLNTGRTEAWKVLSPQGDPWDSVARVLPNGSLLLPAVGIQDEGTFRCRATSRSGKETKSNYRVRVYQIPGKPEIVDPASELMAGVPNKVGTCVSEGGYPAGTLNWLLDGKTLIPDGKGVSVKEETKRHPKTGLFTLHSELMVTPARGGALHPTFSCSFTPGLPRRRALHTAPIQLRVWSEHRGGEGPNVDAVPLKEVQLVVEPEGGAVAPGGTVTLTCEAPAQPPPQIHWIKDGRPLPLPPGPMLLLPEVGPEDQGTYSCVATHPSHGPQESRAVSVTIIETGEEGTTAGSVEGPGLETLALTLGILGGLGTVALLIGVIVWHRRRQRKGQERKVPENQEEEEEERAEL NQPEEPEAAESSTGGPpolyA_signal 1406..1411 polyA_site 1426BASE COUNT 322 a 429 c 440 g 235 t

ORIGIN    1 cggagaagga tggcagcagg ggcagtggtc ggagcctgga tgctagtcctcagtctgggg (SEQ ID NO: 2)   61 gggacagtca cgggggacca aaacatcacagcccggatcg ggaagccact ggtgctgaac  121 tgcaagggag cccccaagaa accaccccagcagctggaat ggaaactgaa cacaggccgg  181 acagaagctt ggaaagtcct gtctccccagggagacccct gggatagcgt ggctcgggtc  241 ctccccaacg gctccctcct cctgccggctgttgggatcc aggatgaggg gactttccgg  301 tgccgggcaa cgagccggag cggaaaggagaccaagtcta actaccgagt ccgagtctat  361 cagattcctg ggaagccaga aattgttgatcctgcctctg aactcatggc tggtgtcccc  421 aataaggtgg ggacatgtgt gtccgaggggggctaccctg cagggactct taactggctc  481 ttggatggga aaactctgat tcctgatggcaaaggagtgt cagtgaagga agagaccaag  541 agacacccaa agacagggct tttcacgctccattcggagc tgatggtgac cccagctcgg  601 ggaggagctc tccaccccac cttctcctgtagcttcaccc ctggccttcc ccggcgccga  661 gccctgcaca cggcccccat ccagctcagggtctggagtg agcaccgagg tggggagggc  721 cccaacgtgg acgctgtgcc actgaaggaagtccagttgg tggtagagcc agaaggggga  781 gcagtagctc ctggtggtac tgtgaccttgacctgtgaag cccccgccca gcccccacct  841 caaatccact ggatcaagga tggcaggcccctgccccttc cccctggccc catgctgctc  901 ctcccagagg tagggcctga ggaccagggaacctacagtt gtgtggccac ccatcccagc  961 catgggcccc aggagagccg tgctgtcagcgtcacgatca tcgaaacagg cgaggagggg 1021 acgactgcag gctctgtgga agggccggggctggaaaccc tagccctgac cctggggatc 1081 ctgggaggcc tggggacagt cgccctgctcattggggtca tcgtgtggca tcgaaggcgg 1141 caacgcaaag gacaggagag gaaggtcccggaaaaccagg aggaggaaga ggaggagaga 1201 gcggaactga accagccaga ggagcccgaggcggcagaga gcagcacagg agggccttga 1261 ggagcccacg gccagacccg atccatcagccccttttctt ttcccacact ctgttctggc 1321 cccagaccag ttctcctctg tataatctccagcccacatc tcccaaactt tcttccacaa 1381 ccagagcctc ccacaaaaag tgatgagtaaacacctgcca cattta//LOCUS HUMRAGE 1391 by mRNA PRI 9 Dec. 1993DEFINITION Human receptor for advanced glycosylation end products (RAGE)mRNA, partial cds.ACCESSION M91211VERSION M91211.1 GI:190845KEYWORDS RAGE; cell surface receptor.SOURCE Homo sapiens cDNA to mRNA.ORGANISM Homo sapiens Eukaryota; Metazoa; Chordata; Craniata;Vertebrata; Euteleostomi; Mammalia; Eutheria; Primates; Catarrhini;Hominidae; Homo.REFERENCE 1 (bases 1 to 1391)AUTHORS Neeper, M., Schmidt, A. M., Brett, J., Yan, S. D., Wang, F.,Pan, Y. C., Elliston, K., Stern, D. and Shaw, A.TITLE Cloning and expression of a cell surface receptor for advancedglycosylation end products of proteinsJOURNAL J. Biol. Chem. 267, 14998-15004 (1992)MEDLINE 92340547REFERENCE 2 (bases 1 to 1391)AUTHORS Shaw, A.TITLE Direct SubmissionJOURNAL Submitted (15-APR-1992) A. Shaw, Department of Cellular andMolecular Biology, Merck Sharp and Dohme Research Laboratories, WestPoint, Pa. 19486 USA FEATURES Location/Qualifiers source 1..1391/organism=“Homo sapiens” /db_xref=“taxon:9606” /tissue_type=“lung” CDS<1..1215 /standard_name=“RAGE” /codon_start=1 /product=“receptor foradvanced glycosylation end products” /protein_id=“AAA03574.1”/db_xref=“GI:190846”/translation=”

(SEQ ID NO: 3) GAAGTAVGAWVLVLSLWGAVVGAQNITARIGEPLVLKCKGAPKKPPQRLEWKLNTGRTEAWKVLSPQGGGPWDSVARVLPNGSLFLPAVGIQDEGIFRCRAMNRNGKETKSNYRVRVYQIPGKPEIVDSASELTAGVPNKVGTCVSEGSYPAGTLSWHLDGKPLVPNEKGVSVKEQTRRHPETGLFTLQSELMVTPARGGDPRPTFSCSFSPGLPRHRALRTAPIQPRVWEPVPLEEVQLVVEPEGGAVAPGGTVTLTCEVPAQPSPQIHWMKDGVPLPLPPSPVLILPEIGPQDQGTYSCVATHSSHGPQESRAVSISIIEPGEEGPTAGSVGGSGLGTLALALGILGGLGTAALLIGVILWQRRQRRGEERKAPENQEEEEERAELNQSEEPEAGESS TGGPpolyA_signal 1368.1373 polyA_site 1391BASE COUNT 305 a 407 c 418 g 261 t

ORIGIN    1 ggggcagccg gaacagcagt tggagcctgg gtgctggtcc tcagtctgtggggggcagta (SEQ ID NO: 4)   61 gtaggtgctc aaaacatcac agcccggattggcgagccac tggtgctgaa gtgtaagggg  121 gcccccaaga aaccacccca gcggctggaatggaaactga acacaggccg gacagaagct  181 tggaaggtcc tgtctcccca gggaggaggcccctgggaca gtgtggctcg tgtccttccc  241 aacggctccc tcttccttcc ggctgtcgggatccaggatg aggggatttt ccggtgcagg  301 gcaatgaaca ggaatggaaa ggagaccaagtccaactacc gagtccgtgt ctaccagatt  361 cctgggaagc cagaaattgt agattctgcctctgaactca cggctggtgt tcccaataag  421 gtggggacat gtgtgtcaga gggaagctaccctgcaggga ctcttagctg gcacttggat  481 gggaagcccc tggtgcctaa tgagaagggagtatctgtga aggaacagac caggagacac  541 cctgagacag ggctcttcac actgcagtcggagctaatgg tgaccccagc ccggggagga  601 gatccccgtc ccaccttctc ctgtagcttcagcccaggcc ttccccgaca ccgggccttg  661 cgcacagccc ccatccagcc ccgtgtctgggagcctgtgc ctctggagga ggtccaattg  721 gtggtggagc cagaaggtgg agcagtagctcctggtggaa ccgtaaccct gacctgtgaa  781 gtccctgccc agccctctcc tcaaatccactggatgaagg atggtgtgcc cttgcccctt  841 ccccccagcc ctgtgctgat cctccctgagatagggcctc aggaccaggg aacctacagc  901 tgtgtggcca cccattccag ccacgggccccaggaaagcc gtgctgtcag catcagcatc  961 atcgaaccag gcgaggaggg gccaactgcaggctctgtgg gaggatcagg gctgggaact 1021 ctagccctgg ccctggggat cctgggaggcctggggacag ccgccctgct cattggggtc 1081 atcttgtggc aaaggcggca acgccgaggagaggagagga aggccccaga aaaccaggag 1141 gaagaggagg agcgtgcaga actgaatcagtcggaggaac ctgaggcagg cgagagtagt 1201 actggagggc cttgaggggc ccacagacagatcccatcca tcagctccct tttctttttc 1261 ccttgaactg ttctggcctc agaccaactctctcctgtat aatctctctc ctgtataacc 1321 ccaccttgcc aagctttctt ctacaaccagagccccccac aatgatgatt aaacacctga 1381 cacatcttgc a//LOCUS MUSRECEP 1348 by mRNA ROD 23-AUG-1994DEFINITION Mouse receptor for advanced glycosylation end products (RAGE)gene, complete cds.ACCESSION L33412VERSION L33412.1 GI:532208KEYWORDS receptor for advanced glycosylation end products.SOURCE Mus musculus (strain BALB/c, sub_species domesticus) (library:lambda gt10) male adult lung cDNA to mRNA.ORGANISM Mus musculus Eukaryota; Metazoa; Chordata; Craniata;Vertebrata; Euteleostomi; Mammalia; Eutheria; Rodentia; Sciurognathi;Muridae; Murinae; Mus.REFERENCE 1 (bases 1 to 1348)AUTHORS Lundh, E. R., Morser, J., McClary, J. and Nagashima, M.TITLE Isolation and characterization of cDNA encoding the murine and rathomologues of the mammalian receptor for advanced glycosylation endproductsJOURNAL UnpublishedCOMMENT On Aug. 24, 1994 this sequence versionreplaced gi:496146.FEATURES Location/Qualifiers source 1..1348/organism=“Mus musculus”/strain=“BALB/c”/sub_species=“domesticus”/db_xref=“taxon:10090”/sex=“male”/tissue_type=“lung”/dev_stage=“adult”/tissue_lib=“lambda gt10” gene6.1217 /gene=“RAGE” CDS6.1217/gene=“RAGE”/codon_start=1/product=“receptor for advancedglycosylation end products”/protein_id=“AAA40040.1”/db_xref=“GI:532209”/translation=”

(SEQ ID NO: 5) MPAGTAARAWVLVLALWGAVAGGQNITARIGEPLVLSCKGAPKKPPQQLEWKLNTGRTEAWKVLSPQGGPWDSVAQILPNGSLLLPATGIVDEGTFRCRATNRRGKEVKSNYRVRVYQIPGKPEIVDPASELTASVPNKVGTCVSEGSYPAGTLSWHLDGKLLIPDGKETLVKEETRRHPETGLFTLRSELTVIPTQGGTTHPTFSCSFSLGLPRRRPLNTAPIQLRVREPGPPEGIQLLVEPEGGIVAPGGTVTLTCAISAQPPPQVHWIKDGAPLPLAPSPVLLLPEVGHADEGTYSCVATHPSHGPQESPPVSIRVTETGDEGPAEGSVGESGLGTLALALGILGGLGVVALLVGAILWRKRQPRREERKAPESQEDEEERAELNQSEEAEMPENGA GGPpolyA_site 1333BASE COUNT 301 a 394 c 404 g 249 t

ORIGIN    1 gcaccatgcc agcggggaca gcagctagag cctgggtgct ggttcttgctctatggggag (SEQ ID NO: 6)   61 ctgtagctgg tggtcagaac atcacagcccggattggaga gccacttgtg ctaagctgta  121 agggggcccc taagaagccg ccccagcagctagaatggaa actgaacaca ggaagaactg  181 aagcttggaa ggtcctctct ccccagggaggcccctggga cagcgtggct caaatcctcc  241 ccaatggttc cctcctcctt ccagccactggaattgtcga tgaggggacg ttccggtgtc  301 gggcaactaa caggcgaggg aaggaggtcaagtccaacta ccgagtccga gtctaccaga  361 ttcctgggaa gccagaaatt gtggatcctgcctctgaact cacagccagt gtccctaata  421 aggtggggac atgtgtgtct gagggaagctaccctgcagg gacccttagc tggcacttag  481 atgggaaact tctgattccc gatggcaaagaaacactcgt gaaggaagag accaggagac  541 accctgagac gggactcttt acactgcggtcagagctgac agtgatcccc acccaaggag  601 gaaccaccca tcctaccttc tcctgcagtttcagcctggg ccttccccgg cgcagacccc  661 tgaacacagc ccctatccaa ctccgagtcagggagcctgg gcctccagag ggcattcagc  721 tgttggttga gcctgaaggt ggaatagtcgctcctggtgg gactgtgacc ttgacctgtg  781 ccatctctgc ccagccccct cctcaggtccactggataaa ggatggtgca cccttgcccc  841 tggctcccag ccctgtgctg ctcctccctgaggtggggca cgcggatgag ggcacctata  901 gctgcgtggc cacccaccct agccacggacctcaggaaag ccctcctgtc agcatcaggg  961 tcacagaaac cggcgatgag gggccagctgaaggctctgt gggtgagtct gggctgggta 1021 cgctagccct ggccttgggg atcctgggaggcctgggagt agtagccctg ctcgtcgggg 1081 ctatcctgtg gcgaaaacga caacccaggcgtgaggagag gaaggccccg gaaagccagg 1141 aggatgagga ggaacgtgca gagctgaatcagtcagagga agcggagatg ccagagaatg 1201 gtgccggggg accgtaagag cacccagatcgagcctgtgt gatggcccta gagcagctcc 1261 cccacattcc atcccaattc ctccttgaggcacttccttc tccaaccaga gcccacatga 1321 tccatgctga gtaaacattt gatacggc//

DEFINITIONS

“DNA sequence” is a linear sequence comprised of any combination of thefour DNA monomers, i.e., nucleotides of adenine, guanine, cytosine andthymine, which codes for genetic information, such as a code for anamino acid, a promoter, a control or a gene product. A specific DNAsequence is one which has a known specific function, e.g., codes for aparticular polypeptide, a particular genetic trait or affects theexpression of a particular phenotype.

“Genotype” is the genetic constitution of an organism.

“Phenotype” is a collection of morphological, physiological andbiochemical traits possessed by a cell or organism that results from theinteraction of the genotype and the environment.

“Phenotypic expression” is the expression of the code of a DNA sequenceor sequences which results in the production of a product, e.g., apolypeptide or protein, or alters the expression of the zygote's or theorganisms natural phenotype.

“Zygote” is a diploid cell having the potential for development into acomplete organism. The zygote can result from parthenogenesis, nucleartransplantation, the merger of two gametes by artificial or naturalfertilization or any other method which creates a diploid cell havingthe potential for development into a complete organism. The origin ofthe zygote can be from either the plant or animal kingdom.

In the practice of any of the methods of the invention or preparation ofany of the pharmaceutical compositions an “therapeutically effectiveamount” is an amount which is capable of alleviating the symptoms of thecognitive disorder of memory or learning in the subject. Accordingly,the effective amount will vary with the subject being treated, as wellas the condition to be treated. For the purposes of this invention, themethods of administration are to include, but are not limited to,administration cutaneously, subcutaneously, intravenously, parenterally,orally, topically, or by aerosol.

By “nervous system-specific” is meant that expression of a nucleic acidsequence occurs substantially in a nervous system tissue (for example,the brain or spinal cord).

Preferably, the expression of the nucleic acid sequence in the nervoussystem tissue represents at least a 5-fold, more preferably, a 10-fold,and, most preferably, a 100-fold increase over expression in non-nervoussystem tissue.

The “non-human animals” of the invention include vertebrates such asrodents, non-human primates, sheep, dog, cow, amphibians, reptiles, etc.Preferred non-human animals are selected from the rodent familyincluding rat and mouse, most preferably mouse.

The “transgenic non-human animals” of the invention are produced byintroducing “transgenes” into the germline of the non-human animal.

ADVANTAGES OF THE PRESENT INVENTION

The transgenic non-human mammals of the present invention will provideinsights with respect to how and where protein interactions occur inAlzheimer's Disease and thus provide more useful models for testing theefficacy of certain drugs in preventing or reducing the onset orprogression of this disease. The transgenic non-human mammals of thepresent invention include recombinant genetic material comprised of anucleic acid sequence encoding RAGE fused to specific promoters capableof expressing the protein in specific tissues such as nerve tissuesgenerally and/or specific types of nerve tissue, e.g., the brain.

As described herein, the current invention provides a number ofadvantages. First, because transgenic animals are generally useful forthe investigation of specific biological processes and for reproducingparticular aspects of human disease, the transgenic animals of theinvention provide an important, reproducible and accurate means forscreening drugs to isolate therapeutic agents. In particular, thetransgenic animals that are described for the first time herein have theadvantage of mimicking the defects observed in patients with Alzheimer'sdisease. Accordingly, the efficacy of a particular therapy may beexamined in the same animal at different disease stages. Importantly,because this invention provides a transgenic animal model of Alzheimer'sdisease with measurable phenotypes, compounds may be screened toidentify those which alleviate this symptom, even absent knowledge ofthe symptom's underlying biological cause.

In addition, although not strictly required for drug screening, theassociated neuro-pathological symptoms exhibited by the transgenicanimal models described herein provide the unique advantage of allowingthe investigation of the etiology of Alzheimer's disease. For example,the appearance of reduced synaptic plasticity or the reduced basalsynaptic transmission may be correlated with the appearance of specificbehavioral impairments within individuals or groups of animals. Inaddition, treatments which are shown to improve memory function may betested for their ability to selectively improve certain pathologicalsymptoms.

Another advantage of this invention is the ease with which thesetransgenic animals are bred to produce identical transgenic progeny. Theanimals of the invention may be generated in sufficient quantity to makethem widely and rapidly available to researchers in this field.

FURTHER DETAILED DESCRIPTION OF THE INVENTION

The present invention also provides for a transgenic nonhuman animalwhose germ or somatic cells contain a nucleic acid molecule whichcomprises: (a) a neuronal tissue specific promoter operatively linked toa DNA sequence encoding a receptor for advanced glycation endproduct(RAGE), introduced into the mammal, or an ancestor thereof, at anembryonic stage.

This transgenic animal may be used in screening methods for compoundswhich would be useful in the treatment of neurological disorders inhumans method for screening compounds for their potential use astherapeutic agents which comprises administering to the transgenicnon-human mammal described herein the compound, in various amounts, andobserving whether the neurological function of the transgenic mammalimproves or not (as determined by, for example, basal synaptictransmission, synaptic plasticity, neuronal stress, et al.).

The neurological disorder may be amnesia, Alzheimer's disease,amyotrophic lateral sclerosis, a brain injury, cerebral senility,chronic peripheral neuropathy, a cognitive disability, a degenerativedisorder associated with learning, Down's Syndrome, dyslexia, electricshock induced amnesia or amnesia. Guillain-Barre syndrome, head trauma,Huntington's disease, a learning disability, a memory deficiency, memoryloss, a mental illness, mental retardation, memory or cognitivedysfunction, multi-infarct dementia and senile dementia, myastheniagravis, a neuromuscular disorder, Parkinson's disease, Pick's disease, areduction in spatial memory retention, senility, or Turret's syndrome.

The compound which is tested in the screening method of the presentinvention may be an organic compound, a nucleic acid, a small molecule,an inorganic compound, a lipid, or a synthetic compound. The mammal maybe a mouse, a sheep, a bovine, a canine, a porcine, or a primate. Theadministration may comprise intralesional, intraperitoneal,intramuscular or intravenous injection; infusion; liposome-mediateddelivery; gene bombardment; topical, nasal, oral, anal, ocular or oticdelivery.

The present invention also provides for a method for alleviatingsymptoms in a subject suffering from a neurological disorder whichcomprises administering to the subject an effective amount of thecompound evaluated by the methods hereinabove in an amount effective totreat the symptoms in the subject suffering from a neurologicaldisorder.

The administration may be intralesional, intraperitoneal, intramuscularor intravenous injection; infusion; liposome-mediated delivery; genebombardment; topical, nasal, oral, anal, ocular or otic delivery.

Pharmaceutical Compositions and Carriers

As used herein, the term “suitable pharmaceutically acceptable carrier”encompasses any of the standard pharmaceutically accepted carriers, suchas phosphate buffered saline solution, water, emulsions such as anoil/water emulsion or a triglyceride emulsion, various types of wettingagents, tablets, coated tablets and capsules. An example of anacceptable triglyceride emulsion useful in intravenous andintraperitoneal administration of the compounds is the triglycerideemulsion commercially known as Intralipid®.

Typically such carriers contain excipients such as starch, milk, sugar,certain types of clay, gelatin, stearic acid, talc, vegetable fats oroils, gums, glycols, or other known excipients. Such carriers may alsoinclude flavor and color additives or other ingredients.

This invention also provides for pharmaceutical compositions includingtherapeutically effective amounts of protein compositions and compoundstogether with suitable diluents, preservatives, solubilizers,emulsifiers, adjuvants and/or carriers useful in treatment of neuronaldegradation due to aging, a learning disability, or a neurologicaldisorder. Such compositions are liquids or lyophilized or otherwisedried formulations and include diluents of various buffer content (e.g.,Tris-HCl., acetate, phosphate), pH and ionic strength, additives such asalbumin or gelatin to prevent absorption to surfaces, detergents (e.g.,Tween 20, Tween 80, Pluronic F68, bile acid salts), solubilizing agents(e.g., glycerol, polyethylene glycerol), anti-oxidants (e.g., ascorbicacid, sodium metabisulfite), preservatives (e.g., Thimerosal, benzylalcohol, parabens), bulking substances or tonicity modifiers (e.g.,lactose, mannitol), covalent attachment of polymers such as polyethyleneglycol to the compound, complexation with metal ions, or incorporationof the compound into or onto particulate preparations of polymericcompounds such as polylactic acid, polglycolic acid, hydrogels, etc, oronto liposomes, micro emulsions, micelles, unilamellar or multi lamellarvesicles, erythrocyte ghosts, or spheroplasts. Such compositions willinfluence the physical state, solubility, stability, rate of in vivorelease, and rate of in vivo clearance of the compound or composition.The choice of compositions will depend on the physical and chemicalproperties of the compound.

Controlled or sustained release compositions include formulation inlipophilic depots (e.g., fatty acids, waxes, oils). Also comprehended bythe invention are particulate compositions coated with polymers (e.g.,poloxamers or poloxamines) and the compound coupled to antibodiesdirected against tissue-specific receptors, ligands or antigens orcoupled to ligands of tissue-specific receptors. Other embodiments ofthe compositions of the invention incorporate particulate formsprotective coatings, protease inhibitors or permeation enhancers forvarious routes of administration, including parenteral, pulmonary, nasaland oral.

Portions of the compound of the invention may be “labeled” byassociation with a detectable marker substance (e.g., radiolabeled with¹²⁵I or biotinylated) to provide reagents useful in detection andquantification of compound or its receptor bearing cells or itsderivatives in solid tissue and fluid samples such as blood, cerebralspinal fluid or urine.

When administered, compounds are often cleared rapidly from thecirculation and may therefore elicit relatively short-livedpharmacological activity. Consequently, frequent injections ofrelatively large doses of bioactive compounds may by required to sustaintherapeutic efficacy. Compounds modified by the covalent attachment ofwater-soluble polymers such as polyethylene glycol, copolymers ofpolyethylene glycol and polypropylene glycol, carboxymethyl cellulose,dextran, polyvinyl alcohol, polyvinylpyrrolidone or polyproline areknown to exhibit substantially longer half-lives in blood followingintravenous injection than do the corresponding unmodified compounds(Abuchowski et al., 1981; Newmark et al., 1982; and Katre et al., 1987).Such modifications may also increase the compound's solubility inaqueous solution, eliminate aggregation, enhance the physical andchemical stability of the compound, and greatly reduce theimmunogenicity and reactivity of the compound. As a result, the desiredin vivo biological activity may be achieved by the administration ofsuch polymer-compound adducts less frequently or in lower doses thanwith the unmodified compound.

Attachment of polyethylene glycol (PEG) to compounds is particularlyuseful because PEG has very low toxicity in mammals (Carpenter et al.,1971). For example, a PEG adduct of adenosine deaminase was approved inthe United States for use in humans for the treatment of severe combinedimmunodeficiency syndrome. A second advantage afforded by theconjugation of PEG is that of effectively reducing the immunogenicityand antigenicity of heterologous compounds. For example, a PEG adduct ofa human protein might be useful for the treatment of disease in othermammalian species without the risk of triggering a severe immuneresponse. The compound of the present invention capable of alleviatingsymptoms of a cognitive disorder of memory or learning may be deliveredin a microencapsulation device so as to reduce or prevent an host immuneresponse against the compound or against cells which may produce thecompound. The compound of the present invention may also be deliveredmicroencapsulated in a membrane, such as a liposome.

Polymers such as PEG may be conveniently attached to one or morereactive amino acid residues in a protein such as the alpha-amino groupof the amino terminal amino acid, the epsilon amino groups of lysineside chains, the sulfhydryl groups of cysteine side chains, the carboxylgroups of aspartyl and glutamyl side chains, the alpha-carboxyl group ofthe carboxy-terminal amino acid, tyrosine side chains, or to activatedderivatives of glycosyl chains attached to certain asparagine, serine orthreonine residues.

Numerous activated forms of PEG suitable for direct reaction withproteins have been described. Useful PEG reagents for reaction withprotein amino groups include active esters of carboxylic acid orcarbonate derivatives, particularly those in which the leaving groupsare N-hydroxysuccinimide, p-nitrophenol, imidazole or1-hydroxy-2-nitrobenzene-4-sulfonate. PEG derivatives containingmaleimido or haloacetyl groups are useful reagents for the modificationof protein free sulfhydryl groups. Likewise, PEG reagents containingamino hydrazine or hydrazide groups are useful for reaction withaldehydes generated by periodate oxidation of carbohydrate groups inproteins.

In one embodiment the compound of the present invention is associatedwith a pharmaceutical carrier which includes a pharmaceuticalcomposition. The pharmaceutical carrier may be a liquid and thepharmaceutical composition would be in the form of a solution. Inanother embodiment, the pharmaceutically acceptable carrier is a solidand the composition is in the form of a powder or tablet. In a furtherembodiment, the pharmaceutical carrier is a gel and the composition isin the form of a suppository or cream. In a further embodiment theactive ingredient may be formulated as a part of a pharmaceuticallyacceptable transdermal patch.

Transgenic Technology and Methods

The following U.S. patents are hereby incorporated by reference: U.S.Pat. No. 6,025,539, IL-5 transgenic mouse; U.S. Pat. No. 6,023,010,Transgenic non-human animals depleted in a mature lymphocytic cell-type;U.S. Pat. No. 6,018,098, In vivo and in vitro model of cutaneousphotoaging; U.S. Pat. No. 6,018,097, Transgenic mice expressing humaninsulin; U.S. Pat. No. 6,008,434, Growth differentiation factor-11transgenic mice; U.S. Pat. No. 6,002,066; H2-M modified transgenic mice;U.S. Pat. No. 5,994,618, Growth differentiation factor-8 transgenicmice; U.S. Pat. No. 5,986,171, Method for examining neurovirulence ofpolio virus, U.S. Pat. No. 5,981,830, Knockout mice and their progenywith a disrupted hepsin gene; U.S. Pat. No. 5,981,829, .DELTA.Nur77transgenic mouse; U.S. Pat. No. 5,936,138; Gene encoding mutant L3T4protein which facilitates HIV infection and transgenic mouse expressingsuch protein; U.S. Pat. No. 5,912,411, Mice transgenic for atetracycline-inducible transcriptional activator; U.S. Pat. No.5,894,078, Transgenic mouse expressing C-100 app.

The methods used for generating transgenic mice are well known to one ofskill in the art. For example, one may use the manual entitled“Manipulating the Mouse Embryo” by Brigid Hogan et al. (Ed. Cold SpringHarbor Laboratory) 1986.

See for example, Leder and Stewart, U.S. Pat. No. 4,736,866 for methodsfor the production of a transgenic mouse.

For sometime it has been known that it is possible to carry out thegenetic transformation of a zygote (and the embryo and mature organismwhich result therefrom) by the placing or insertion of exogenous geneticmaterial into the nucleus of the zygote or to any nucleic geneticmaterial which ultimately forms a part of the nucleus of the zygote. Thegenotype of the zygote and the organism which results from a zygote willinclude the genotype of the exogenous genetic material. Additionally,the inclusion of exogenous genetic material in the zygote will result ina phenotype expression of the exogenous genetic material.

The genotype of the exogenous genetic material is expressed upon thecellular division of the zygote. However, the phenotype expression,e.g., the production of a protein product or products of the exogenousgenetic material, or alterations of the zygote's or organism's naturalphenotype, will occur at that point of the zygote's or organism'sdevelopment during which the particular exogenous genetic material isactive. Alterations of the expression of the phenotype include anenhancement or diminution in the expression of a phenotype or analteration in the promotion and/or control of a phenotype, including theaddition of a new promoter and/or controller or supplementation of anexisting promoter and/or controller of the phenotype.

The genetic transformation of various types of organisms is disclosedand described in detail in U.S. Pat. No. 4,873,191, issued Oct. 10,1989, which is incorporated herein by reference to disclose methods ofproducing transgenic organisms. The genetic transformation of organismscan be used as an in vivo analysis of gene expression duringdifferentiation and in the elimination or diminution of genetic diseasesby either gene therapy or by using a transgenic non-human mammal as amodel system of a human disease. This model system can be used to testputative drugs for their potential therapeutic value in humans.

The exogenous genetic material can be placed in the nucleus of a matureegg. It is preferred that the egg be in a fertilized or activated (byparthenogenesis) state. After the addition of the exogenous geneticmaterial, a complementary haploid set of chromosomes (e.g., a sperm cellor polar body) is added to enable the formation of a zygote. The zygoteis allowed to develop into an organism such as by implanting it in apseudopregnant female. The resulting organism is analyzed for theintegration of the exogenous genetic material. If positive integrationis determined, the organism, can be used for the in vivo analysis of thegene expression, which expression is believed to be related to aparticular genetic disease.

Attempts have been made to study a number of different types of geneticdiseases utilizing such transgenic animals. Attempts related to studyingAlzheimer's disease are disclosed within published PCT applicationWO89/06689 and PCT application WO89/06693, both published on Jul. 27,1989, which published applications are incorporated herein by referenceto disclose genetic sequences coding for Alzheimer's .beta.-amyloidprotein and the incorporation of such sequences into the genome oftransgenic animals.

Embryonal target cells at various developmental stages can be used tointroduce transgenes. Different methods are used depending on the stageof development of the embryonal target cell. The zygote is the besttarget for micro-injection. In the mouse, the male pronucleus reachesthe size of approximately 20 micrometers in diameter which allowsreproducible injection of 1-2 pl of DNA solution. The use of zygotes asa target for gene transfer has a major advantage in that in most casesthe injected DNA will be incorporated into the host gene before thefirst cleavage (Brinster, et al. (1985) Proc. Natl. Acad. Sci. U.S.A.82, 4438-4442). As a consequence, all cells of the transgenic non-humananimal will carry the incorporated transgene. This will in general alsobe reflected in the efficient transmission of the transgene to offspringof the founder since 50% of the germ cells will harbor the transgene.Microinjection of zygotes is the preferred method for incorporatingtransgenes in practicing the invention.

Retroviral infection can also be used to introduce transgene into anon-human animal. The developing non-human embryo can be cultured invitro to the blastocyst stage. During this time, the blastomeres can betargets for retroviral infection (Jaenich, R. (1976) Proc. Natl. Acad.Sci U.S.A. 73, 1260-1264). Efficient infection of the blastomeres isobtained by enzymatic treatment to remove the zona pellucida (Hogan, etal. (1986) in Manipulating the Mouse Embryo, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.). The viral vector systemused to introduce the transgene is typically a replication-defectiveretrovirus carrying the transgene (Jahner, et al. (1985) Proc. Natl.Acad. Sci. U.S.A. 82, 6927-6931; Van der Putten, et al. (1985) Proc.Natl. Acad. Sci U.S.A. 82, 6148-6152). Transfection is easily andefficiently obtained by culturing the blastomeres on a monolayer ofvirus-producing cells (Van der Putten, supra; Stewart, et al. (1987)EMBO J. 6, 383-388). Alternatively, infection can be performed at alater stage. Virus or virus-producing cells can be injected into theblastocoele (Jahner, D., et al. (1982) Nature 298, 623-628). Most of thefounders will be mosaic for the transgene since incorporation occursonly in a subset of the cells which formed the transgenic non-humananimal. Further, the founder may contain various retroviral insertionsof the transgene at different positions in the genome which generallywill segregate in the offspring. In addition, it is also possible tointroduce transgenes into the germ line, albeit with low efficiency, byintrauterine retroviral infection of the midgestation embryo (Jahner, D.et al. (1982) supra).

A third type of target cell for transgene introduction is the embryonalstem cell (ES). ES cells are obtained from pre-implantation embryoscultured in vitro and fused with embryos (Evans, M. J., et al. (1981)Nature 292, 154-156; Bradley, M. O., et al. (1984) Nature 309, 255-258;Gossler, et al. (1986) Proc. Natl. Acad. Sci U.S.A. 83, 9065-9069; andRobertson, et al. (1986) Nature 322, 445-448). Transgenes can beefficiently introduced into the ES cells by DNA transfection or byretrovirus-mediated transduction. Such transformed ES cells canthereafter be combined with blastocysts from a non-human animal. The EScells thereafter colonize the embryo and contribute to the germ line ofthe resulting chimeric animal. For review see Jaenisch, R. (1988)Science 240, 1468-1474.

As used herein, a “transgene” is a DNA sequence introduced into thegermline of a non-human animal by way of human intervention such as byway of the above described methods.

The disclosures of publications referenced in this application in theirentireties are hereby incorporated by reference into this application inorder to more fully describe the state of the art as known to thoseskilled therein as of the date of the invention described and claimedherein.

This invention is illustrated in the Experimental Details section whichfollows. These sections are set forth to aid in an understanding of theinvention but are not intended to, and should not be construed to, limitin any way the invention as set forth in the claims which followthereafter.

EXPERIMENTAL DETAILS Example 1 Generation of Transgenic Mice withTargeted Overexpression of Receptor for Advanced Glycation Endproducts(RAGE) in Neurons

This paper describes a means of making transgenic mice with targetedoverexpression of RAGE in neurons using the PDGF B-chain promoter andthe cDNA for human full-length RAGE. The mice, termed Tg PD-RAGE, whichhave been produced provide a model system for determining theconsequences of heightened RAGE expression in neurons, and could serveas an important model system to test RAGE blockers, either inhibitors ofligand-receptor interaction or inhibitors of RAGE-dependentintracellular signalling. Cross-breeding of Tg PD-RAGE with otheranimals, such as those expressing a transgene causing overexpression ofmutant amyloid precursor protein (resulting in increased production ofamyloid-beta peptide) provide a model system to assess the effects ofRAGE in an Aβ-rich environment in the brain relevant to Alzheimer'sdisease. In addition, isolation and culture of embryonic neurons from TgPD-RAGE mice allows study of the consequences of increased levels ofRAGE in vitro in actual neurons. These are several examples of how TgPD-RAGE mice can be used to assess the contribution of RAGE, analyzedaccording to in vitro and in vivo systems, to situations potentiallyrelevant to human disease.

Introduction

Receptor for Advanced Glycation Endproducts (RAGE) is a multiligandmember of the immunoglobulin superfamily of cell surface molecules¹. Inthe central nervous system, RAGE is present at high levels in earlydevelopment, but then its expression falls off with maturity^(2,3).However, with development of a pathology in the central nervous system,such as Alzheimer's disease (AD), RAGE expression increases to highlevels⁴. Similarly, in murine models of stroke RAGE expression alsoincreases. This suggests that RAGE may participate in the host response,though it is not clear if its participation would be favorable ordeleterious to the outcome.

There are several ways to dissect the contribution of RAGE inphysiologic and pathophysiologic settings. One method which we have usedextensively is administration of soluble RAGE, the extracellular domain,which serve as a decoy for ligands seeking out cell surface receptor,and anti-RAGE IgG⁵⁻⁷. However these reagents are principally useful intissues served by the systemic circulation which provides the means fordelivery. Tissues behind the blood-brain barrier, such as neurons (e.g.,the entire central nervous-system) pose a more difficult problem sincemacromolecules do not have ready access under most conditions. For thisreason, we sought to create genetic models to dissect the contributionof RAGE. In this context, there are three general approaches:

Targeted over-expression of wild-type RAGE wherein the transgenicnon-human animal would exhibit exaggerated effects of RAGE in theparticular tissue/cell under study (the RAGE is overexpressed in atissue specific manner.

Targeted overexpression of a mutant form of the receptor in which theRAGE cytosolic tail has been deleted. This form of the moleculefunctions as a dominant-negative, thereby blocking the effects of ligandengagement of wild-type RAGE.

Deletion of the RAGE gene by homologous recombination (this can also becarried out using conditional strategies to achieve tissue-specificknockout. (Also termed, a RAGE knockout transgenic non-human animal.)

This report describes the generation of mice with targetedoverexpression of human RAGE in neurons using the PDGF B-chain promoter,termed Tg PD-RAGE mice. In addition, we describe the effect of stroke onTg PD-RAGE mice, and the results of crossing Tg PD-RAGE mice withanimals overexpressing a mutant form of amyloid precursor protein (APP)which causes increased production of amyloid-beta peptide (Aβ).

Methods

Construction of the transgene and making the transgenic (Tg) mice. Theplatelet-derived growth factor (PDGF) B-chain.

promoter was used to drive overexpression of RAGE in neurons of thecentral nervous system of transgenic (Tg) mice⁸. Transgene constructswere prepared using a previously described vector^(9,10). Briefly, theCMV, immediate/early promoter was excised from the commercial expressionvector pCI (Promega, Madison Wis.), and replaced with an oligonucleotidepolylinker. The PDGF B-chain promoter fragment was mobilized as an XbaIfragment⁸ and cloned into a unique SpeI site designed within thesynthetic linker. The full-length human RAGE cDNA was inserted into theNotI site of the original polylinker. A schematic representation of thisconstruct is shown in FIG. 1A (upper panel). An ˜3 kb fragmentcontaining the promoter, cDNA and required other sequences was thenexcised from the plasmid backbone as a PvuI fragment (FIG. 1B, lowerpanel) and microinjected mouse B6CBAF₁/J oocytes. The latter wereimplanted into pseudopregnant females and mated with B6CBAF₁/J malesresulting in the generation of independent founders. Breeding of thesemice demonstrated germ-line transmission and was used to produce linesof animals termed Tg PD-RAGE.

Founders were initially identified by Southern blotting performed on DNAextracted from mouse tails. DNA was digested, run on agarose gels, andhybridized with ³²P-labelled cDNA for human RAGE. Tails were digestedwith proteinase K (500 μg/ml) in digestion buffer (50 mM Tris, pH 8.0,100 mM EDTA, 0.5% SDS) at 55° C. for overnight. Then, purified DNA wascleaved with EcoRI overnight at 37° C. Labelling of the probe was doneusing the Stratagene's Probe Labeling Kit™. Autoradiography was thenperformed.

Subsequent screening of progeny was by PCR using the following primers:5′-AGCGGCTGGAATGGAAACTGAACA-3′ (SEQ ID NO:7) and3′-GAAGGGGCAAGGGCACACCATC-5′ (SEQ ID NO:8). Total RNA was isolated usingTrizol®, and RT-PCR was performed with the following thermocyclingparameters: 30 sec for each cycle consisting of incubations at 95° C.for 20 sec, 57° C. for 30 sec and 72° C. for 1 sec for a total of 35cycles. Products were analyzed by agarose gel electrophoresis andvisualized by ethidium bromide staining under ultraviolet illumination.The size of the RAGE amplicon with these primers corresponded to 701 bp.

Northern and immunoblotting utilized the same procedures as above,except that tissue was homogenized in the presence of Trizol (RNA) or inhomogenization buffer (Tris/HCl, 10 mM, pH 7.4; NaCl, 100 mM;phenylmethylsulfonylfluoride, 100 μg/ml; EDTA, 1 mM; aprotonin, 1μg/ml). Note that immunoblotting and immunostaining of brain tissue fromTg PD-ABAD mice used anti-human RAGE IgG⁷.

Characterization of RAGE expression in Tg PD-RAGE mice. Northernanalysis was performed on total RNA isolated from cerebral cortex,hippocampus and cerebellum. RNA was isolated using Trizol® followed byelectrophoresis on 0.8% agarose gels (30 μg was applied to each lane),transfer to nitrocellulose membranes, and hybridization with³²P-labelled human RAGE cDNA. The RAGE cDNA was labelled by as above.

Western blotting was performed on protein extracts of brain subregions.Proteins were extracted from minced pieces of brains by exposing thetissue to lysis buffer (Tris/HCl, 20 mM; pH 7.4; Triton X-100, 1%;phenylmethylsulfonylfluoride, 2 mM; EDTA, 1 mM, aprotonin, 10 μg/ml;leupeptin, 10 μg/ml) using a ratio of 1 ml of buffer per 0.5 gm oftissue. Extracts were then boiled in reducing SDS-sample buffer, andapplied to SDS-PAGE according to Laemmli¹¹.

Immunostaining was performed on paraformaldehyde (4%)-fixed, paraffinembedded sections (6 μm) of mouse brains prepared according to standardmethods^(4,7). The sections were deparaffinized and dehydrated, and thenstained with rabbit anti-human RAGE IgG (50 μg/ml) followed by goatanti-rabbit biotin-conjugated IgG and ExtrAvidin-conjugated withalkaline phosphatase (Biotin ExtrAvidin® kit; Sigma, St. Louis Mo.).Preparation of Anti-Rage IgG, Using Recombinant Soluble Rage as theimmunogen, has been described^(4,7).

Induction of stroke in Tg PD-RAGE mice. Functional consequences ofoverexpression of RAGE were first assessed in response to ischemicstress, the transient middle cerebral artery occlusion model. Murinestroke model Mice (C57BL6/J, male) were subjected to stroke according topreviously published procedures¹². Following anesthesia, the carotidartery was accessed using the operative approach previously described indetail¹³, including division/coagulation of the occipital andpterygopalatine arteries to obtain improved visualization and vascularaccess. A nylon suture was then introduced into the common carotidartery, and threaded up the internal carotid artery to occlude theorigin of the right middle cerebral artery (MCA). Nylon (polyamide)suture material was obtained from United States Surgical Corporation(Norwalk, Conn.), and consisted of 5.0 nylon/13 mm length for 27-36 gmice, and 6.0 nylon/12 mm length for 22-26 g mice. After 45 minutes ofocclusion, the suture was withdrawn to achieve a reperfused model ofstroke. Although no vessels were tied off after the suture was removed,the external carotid arterial stump was cauterized to prevent frankhemorrhage.

Measurements of relative cerebral blood flow were obtained as previouslyreported¹²⁻¹⁵ using a straight laser doppler flow probe placed 2 mmposterior to the bregma, and 6 mm to each side of midline using astereotactic micromanipulator, keeping the angle of the probeperpendicular to the cortical surface. These cerebral blood flowmeasurements, expressed as the ratio of ipsilateral to contralateralblood flow, were obtained at baseline, and immediately prior to MCAocclusion, 45 minutes after MCA occlusion, and at several time pointsafter withdrawal of the occluding suture.

Measurement of Cerebral Infarction Volumes: After 24 hours, animals wereeuthanized and their brains rapidly harvested. Infarct volumes weredetermined by staining serial cerebral sections with triphenyltetrazolium chloride and performing computer-based planimetry of thenegatively (infarcted) staining areas to calculate infarct volume (usingNIH image software).

Neurological Exam: Prior to giving anesthesia, mice were examined forneurological deficit 23 h after reperfusion using a four-tiered gradingsystem: a score of 1 was given if the animal demonstrated normalspontaneous movements; a score of 2 was given if the animal was noted tobe turning towards the ipsilateral side; a score of 3 was given if theanimal was observed to spin longitudinally (clockwise when viewed fromthe tail); and, a score of 4 was given if the animal was unresponsive tonoxious stimuli. This scoring system has been previously described inmice¹²⁻¹⁴, and is based upon similar scoring systems used in rats¹⁶.Immunostaining of cerebral cortex following induction of stroke inwild-type mice was performed as described above using a rabbitpolyclonal antibody made using purified recombinant murine ABAD as theimmunogen. Quantitation of microscopic images was accomplished with theUniversal Imaging System.

Cross-breeding of Tg PD-RAGE mice with Tg hAPP mice. Tg miceoverexpressing an alternatively spliced hAPP minigene that encodeshAPP695, hAPP751, and hAPP770 bearing mutations linked to familial AD(V717F, K670M/N671L) have been produced by Dr. Lennart Mucke¹⁷, andprovided to us for use in cross-breeding studies with Tg PD-RAGE mice.In these mice, expression of the transgene is also driven by the PDGFB-chain promoter. Cross-breeding was performed and double-transgenicmice expressing both hAPP and PD-RAGE transgenes were identified withspecific primers. The primers for the hAPP transgene were:5′-GACAAGTATCTCGAGACACCTGGGGATGAG-3′ (SEQ ID NO:9) and3′-AAAGAACTTGTAGGTTGGATTTTCGTACC-5′ (SEQ ID NO:10). PCR conditions forthe amplifying the hAPP transgene were the same as those describedabove, and the size of the amplicon was 1169 bp.

Characterization of Tg PD-RAGE/hAPP mice. Mice were anesthetizedaccording to standard procedures and then flush-perfused transcardiallywith solution containing NaCl (0.9%). Brains will then be rapidlyremoved and divided sagitally. One hemibrain was postfixed inphosphate-buffered saline (PBS) containing paraformaldehyde (4%; pH 7.4)at 4° C. for 48 hrs prior to vibratome sectioning. Hemibrains werestored in cryoprotectant medium (phosphate-buffered saline containingglycerin and ethylene glycol) until sectioning. This portion of thebrain was employed for studies of neuronal integrity and degeneration.There is ample evidence that loss of synaptophysin immunoreactivity inpresynaptic terminals is associated with AD brain, a marker whichcorrelates with extent of cognitive impairments¹⁸⁻²³. Additionally,immunoreactivity for Microtubule-associated protein (MAP)-2 was examinedas a marker of neuronal cell bodies and dendrites, as a significantdecrease of MAP-2 immunoreactive dendrites has been observed forexample, in brains of patients with neurodegeneration subsequent toHIV-1 encephalitis²⁴.

Hemibrains were subjected to sagittal sectioning employing a LeicaVibratome® 1000E. Sections, 40 μm thick, were prepared and collectedinto the wells of 12-well tissue culture plates in cryoprotectant mediumand stored at −20° C. until immunostaining was performed. Two sectionsper mouse were randomly selected for further study, based on fullintegrity of the sample, i.e., clearly delineated neocortex, and CA1,CA2 and CA3 were completely intact. Prior to immunohistochemistry,free-floating sections were placed individually in wells of 24-welltissue culture plates and washed twice in phosphate-buffered saline(PBS; pH 7.4; containing calcium/magnesium). Sections were thenpermeabilized in PBS containing Triton X-100 (0.2) for 20 min at roomtemperature. Sections were stained with antibodies to perform assessmentof neuronal integrity. Anti-synaptophysin IgG (Boehringer) was employedas a marker of presynaptic terminals. Anti-MAP-2 IgG (Boehringer) wasemployed as a marker of neuronal cell bodies/dendrites. The appropriatenonimmune IgG was employed as a control (Boehringer Mannheim). Afterappropriate blocking steps, primary antibodies were incubated withsections and then washed in PBS. FITC-labeled secondary antibodies(Vector system; ABC) were employed to visualize sites of primaryantibody binding. After washing, sections were mounted using Vectashieldon glass slides and then coverslips placed atop the sections. Sectionswill then be kept in the dark at 4° C., for no more than two weeks priorto analysis.

Semiquantitative evaluation of neuronal integrity was performed usinglaser scanning confocal microscopy. Neuronal integrity was assessed inthe neocortex and pyramidal cell layer of the hippocampus (CA1 subfield)in six sections per mouse (two for each antibody marker). For eachmouse, 4-8 confocal images (3-4/section) of the neocortex, and 2-4confocal images (1-2/section) of the hippocampal CA1 subfield, eachcovering an area of 210×140 μm, were obtained. Under 60×, oil immersion,the sample was focused and iris and gain levels adjusted to obtainimages with a pixel intensity within a linear range. Each final imagewas processed sequentially in Lasersharp. Digitized images were thentransferred to a Macintosh computer using Adobe Photoshop, JPEGcompression and analyzed with NIH Image. The area of the neuropiloccupied by MAP-2-labeled dendrites or by synaptophysin-labeledpresynaptic terminals was quantified and expressed as a percentage ofthe total image area as described²⁴. Final analysis of digitized imagesfor area neuropil occupied was determined by two independentinvestigators. Mean±standard deviation is reported for each section.Control sections were studied under conditions in which primary antibodywas omitted, and no signal was observed with secondary antibody alone.

Immunostaining of mouse brain for other markers employed commerciallyavailable goat antibody to murine macrophage-colony stimulating factor(goat anti-M-CSF IgG; 10 μg/ml; Santa Cruz), rabbit antibody toactivated caspase 3 (50 μg/ml; PharMingen), and mouse monoclonalantibody to phosphorylated tau (AT8; 10 μg/ml; Immunogenetics). In eachcase, the immunostaining protocol used standard techniques according tothe manufacturer's instructions. Rabbit anti-murine Interleukin (IL) 6IgG was provided by Dr. Gerald Fuller (University of Alabama MedicalCenter, Birmingham) and has been used in previous studies²⁵. Sectionswere incubated with primary antibodies overnight at 4° C., followed byblocking with appropriate antisera and addition of biotin-conjugatedgoat anti-rabbit, goat anti-mouse or mouse anti-goat IgG (1:25 dilution)for 30 min at 37° C. Then ExtrAvidin® conjugated to peroxidase or toalkaline phosphatase (1:25 dilution) was added for 25 min at 37° C.Slides were then washed and developed with 3-amino-9-ethyl carbazole orfast red. Sections were viewed in an Olympus microscope, and images werequantified using the Universal imaging system.

Activation of the transcription factor NF-kB was studied in brains ofmice and in neurons cultured from this tissue (see below). Nuclearextracts were prepared according to the method of Dignam et al.²⁶, andwere incubated with ³²P-labelled double stranded consensusoligonucleotide probe for NF-kB (Santa Cruz) followed by polyacrylamidegel electrophoresis and autoradiography. These methods have beendescribed previously using brain tissue and cultured cells as thesamples for preparation of nuclear extracts⁴.

Isolation and characterization of neurons from Tg PD-RAGE mice. Brainsof E16-18 mouse embryos were processed by a modification of a previouslydescribed method²⁷. In brief, Embryos were washed in ethanol (75%),transferred to a dish with sterile phosphate-buffered saline (PBS) at 4°C. under a tissue culture hood. Two embryos were dissected at a timeimmersed in Neurobasal Medium (GIBCO) with NaCl (22 mM), NaHCO₃ (4.4mM), penicillin (50 units/ml) and streptomycin (50 μg/ml). The embryotail was removed and analyzed by PCR to determine genotype (as above).Cerebral cortex was dissected free from the cerebellum and brainstem,sliced into 1 mm pieces, and transferred to 1.5 ml eppendorf tubes inthe above medium. The tube was centrifuged at 400 rpm, the pellet waswashed in PBS, and resuspended in PBS. Then, trypsin (0.25%; 0.5 ml) andDNAse (250 units/ml) were added and the incubation was continued for 15min at 37° C. with gentle shaking. The mixture was decanted, washedtwice in PBS by inverting the tube and gently spinning at 400 rpm for 5min. Neurons were cultured in growth medium (Neurobasal Medium with B27,2%, L-glutamine, 2 mM, penicillin, 50 units/ml, streptomycin, 50 μg/ml)in wells coated with poly-L-lysine. Neurons were identifiedimmunocytochemically using antibody to neurofilament (Sigma) and themethods for immunostaining described above.

Cultures of neurons were exposed to preformed Aβ(1-42) fibrils for 5 hrsat 37° C. Aβ(1-42) synthetic peptide was purchased from QCB, and wasincubated for 5 days at 37° C. to allow fibril formation to occur. Thepresence of fibrils was confirmed by electron microscopy. Fibrilpreparations were then frozen (−20° C.) until use. Just prior to anexperiment, fibril preparations were thawed, vortexed, and then added toculture medium to a final concentration of 0.5 μM for 5 hrs (NF-kBactivation) or 30-40 hrs (detection of activated caspase 3) at 37°.Nuclear extracts were prepared and EMSA was performed as above. Whereindicated, anti-RAGE IgG was added during incubation of Aβ fibrils withthe cells.

Caspase 3 activity was studied using a kit from Clontech.

Results

Identification of Tg PD-RAGE mice. Southern analysis identifying threeTg PD-RAGE founders is shown in FIG. 2. These mice were used to generatelines of Tg PD-RAGE mice in whom the progeny were identified by PCRusing the primers described in the methods section. An example of PCRdetection of positive founders, versus negative non-Tg animals is shownin FIG. 3.

Characterization of Tg PD-RAGE mice. Expression of the transgene in thebrain was determined by Northern analysis of total RNA extracted fromcerebral cortex with ³²P-labelled cDNA for human RAGE (FIG. 4A). Anintense band was observed in Tg PD-RAGE mice, but not in nontransgeniclittermate controls. Similarly Western analysis was performed on proteinextracts of cerebral cortex from transgenic mice using antibody to theextracellular domain of recombinant human RAGE. Again, a strong band ofimmunoreactivity migrating above the 46 kDa molecular weight markerconfirmed the presence of high levels of RAGE antigen in brains of TgPD-RAGE mice compared with nontransgenic littermate controls (FIG. 4B).Immunoblotting was then performed on brain subregions, includingcerebral cortex, hippocampus and cerebellum, with anti-RAGE IgG (FIG.5). In Tg PD-RAGE mice, intense immunoreactive bands were seen incerebral cortex and hippocampus, compared with lower levels of transgeneexpression in the cerebellum. Immunostaining with anti-RAGE IgGconfirmed that the increased levels of RAGE in Tg PD-RAGE animals werein neurons (FIGS. 6A-B). These data indicate that Tg PD-RAGE miceprovide a model system in which neurons of the brain, especiallycerebral cortex and hippocampus express high levels of RAGE.

To examine consequences of enhanced neuronal RAGE expression forischemic stress experiments were performed in a murine model oftransient occlusion of the middle cerebral artery (FIG. 7). Tg PD-RAGEmice, as well as nonTg littermate controls weighed ≈126 grams and were≈10 wks old. Neurologic deficit score, evaluated at the 24 hr point, wasreduced in Tg PD-RAGE mice compared with nonTg littermates. The volumeof infarcted cerebral tissue was reduced by ˜50% in Tg mice (FIG. 7;p<0.05).

Characterization of Tg PD-RAGE/hAPP mice. The increased expression ofRAGE brain, compared with age-matched non-demented brain, suggested thatthe receptor might be associated with AD pathology. Consistent with thishypothesis, our pilot studies with Tg hAPP mice displayed increasedlevels of RAGE in cerebral cortex by 4 months of age, which is prior toplaque formation. Tg hAPP mice are especially useful for studies toassess the effect of introduction of the PD-RAGE transgene since theyhave been characterized in previous studies with respect toneuropathologic and electrophysiologic properties.

If RAGE-Aβ interaction promoted neuronal stress, we reasoned thatexpression of high levels of RAGE at early times in the brains ofanimals expressing the hAPP transgene might result in magnified cellstress and cytotoxicity (the transgenic model introduces higher levelsof RAGE expression than were present in Tg hAPP mice alone, and thus,potentially represents a model of exaggerated effects of RAGE due tooverexpression of the receptor). Cross-breeding studies were performedand double transgenic mice were identified by PCR using primers specificfor the PD-RAGE transgene and the hAPP transgene. Results of arepresentative PCR analysis are shown in FIG. 8 demonstrating ampliconsfor both the PD-RAGE and hAPP genes in the double-transgenic animalsversus single transgenics and nontransgenic littermate controls. Thedouble transgenic mice have been termed Tg PD-RAGE/hAPP mice.

Double transgenic mice were observed for three-four months and evidenceof neuronal stress was then analyzed by studying expression of M-CSF(FIGS. 9A, 9B1-B3, 9C) and IL-6 (FIGS. 10A, 10B1-B3, 10C), andactivation of NF-kB (FIG. 11). Northern analysis of cerebral cortexshowed increased levels of transcripts for M-CSF in Tg PD-RAGE/hAPP micecompared with single transgenics (Tg hAPP and Tg PD-RAGE) and nonTglittermate controls (FIG. 9A). Immunostaining with anti-M-CSF IgGconfirmed that the increase in M-CSF antigen was predominately inneurons, and that double-transgenics showed elevated levels of antigencompared with the other groups (FIG. 9B1, 9B2, 9B3 and 9C). Similarresults were observed with respect to increased expression of IL-6transcripts and antigen (FIG. 10A, 10B1-B3, 10C)). Since activation ofNF-kB is associated with cellular stress responses and can underlieexpression of M-CSF and IL-6, we analyzed nuclear translocation of NF-kBin nuclear extracts prepared from brains of the transgenic mice by EMSA(FIG. 11). Analysis of double transgenic mice invariably displayed astrong gel shift band, whose intensity varied somewhat in the differentanimals, whereas weak/absent bands were seen in the control group andsingle transgenics.

These data were consistent with increased neuronal stress in TgPD-RAGE/hAPP mice, but did not indicate whether the outcome of thisstress would be neuroprotection or neurotoxicity. To analyze thissituation neuropathologic studies were performed, and study of markersmore clearly associated with toxicity, such as activated caspase 3, wasundertaken. Immunostaining of hippocampus (lacunosum moleculare layer)from double transgenic mice (age 3-4 months) with antibody tosynaptophysin demonstrated a reduction in the area of neuropil occupiedby synaptophysin-labeled presynaptic terminals (FIG. 12). Similarstudies with antibody to MAP-2 showed a reduction in the area ofneuropil occupied by MAP-2-labeled dendrites (FIG. 13). Althoughstereologic studies will be required to determine actual neuronal loss,these results are consistent with evidence of neurotoxicity. Consistentwith this impression, analysis of older double transgenic mice (8-9months of age) showed increased staining with an antibody selective forthe activated form of caspase 3 (FIGS. 14A1-4, 14B) and phosphorylatedtau (AT8) (FIGS. 15A1-A4, 15B) in brains of double transgenic micecompared with the other groups.

Culture of neurons from Tg PD-RAGE mice. Neuronal cultures were madefrom the cerebral cortex from E16 mouse embryos. These cultureswere >90% neurons based on staining with anti-neurofilament antibody.Cultured neurons displayed high levels of RAGE expression based onimmunoblotting (FIG. 16). Such neuronal cultures were incubated withpreformed Aβ(1-40) fibrils (0.5 μM) for 2 hrs, and then nuclear extractswere prepared for EMSA using consensus probe for NF-kB. A gel shift bandwas observed in neuronal cultures from Tg PD-RAGE mice, but such a bandwas either not seen or present at much reduced intensity with samplesfrom nontransgenic littermate control mice (FIG. 17A). That this was dueto Aβ-RAGE interaction was confirmed by the dose-dependent inhibitoryeffect of anti-RAGE IgG, but not nonimmune IgG (FIG. 17B). To determinewhether neurons overexpressing RAGE exposed to Aβ fibrils were beingforced down a pathway of enhanced toxicity evidence of activated caspase3 was sought. After 30-40 hrs of exposing Aβ fibrils to neurons from TgPD-RAGE mice, increased caspase 3 activity was detected (FIG. 18). Incontrast, under these conditions, neurons form nonTg littermates did notdisplay increased caspase 3 activity.

Discussion

We have described the generation of Tg PD-RAGE mice which displayincreased expression of full-length RAGE in neurons. The use of thesemice to analyze the contribution of RAGE to cellular responses in vitroand in vivo can be summarized according to three general categories:

1) analysis of neurons cultured from embryos of Tg PD-RAGE mice. Sinceneurons are nondividing cells and can only be transfected successfullywith viral-based systems (which can alter cellular propertiesthemselves), the cultured neurons from Tg PD-RAGE mice provide an uniquesystem in which neurons overexpress human RAGE. These cells can be usedto analyze the consequences of RAGE-ligand interaction for neuronalfunction, as shown by the activation of NF-kB and activation of caspase3 in the above studies.

2) Tg PD-RAGE mice can be used to directly assess the effect of RAGEoverexpression in settings such as, but not limited to, stroke(described above), viral/bacterial infections, other models of braininflammation (such as experimental autoimmune encephalitis) etc.

3) Tg PD-RAGE mice can be crossbred with other transgenic animals, suchas Tg hAPP to determine the consequences of RAGE overexpression in anenvironment in which the other transgene creates an unique environmentin the brain. For example, the Tg hAPP mouse results in increased levelsof Aβ. In this setting, the consequences of increased levels of Aβ inthe context of neurons bearing elevated levels of RAGE can be studied.Another example would be cross-breeding of Tg PD-RAGE mice with micedeficient in the gene for apolipoprotein E²⁸, resulting in a model ofaccelerated atherosclerosis potentially with an exaggerated effect ofRAGE.

In each case, the in vitro and in vivo systems based on Tg PD-RAGE miceor cells derived from them are ideal for studying RAGE inhibitors, aswell as for dissecting contributions of RAGE tophysiologic/pathophysiologic outcomes.

Example 2 Induction of Transient Middle Cerebral Artery in RAGETransgenic Mice and Use of this Transgenic Mouse Model for Stroke inHumans

Methods:

Induction of Transient Middle Cerebral Artery Occlusion in the Mouse

Induction of stroke in Tg PD-RAGE mice. Functional consequences ofoverexpression of RAGE were first assessed in response to ischemicstress, the transient middle cerebral artery occlusion model. Murinestroke model Mice (C57BL6/J, male) were subjected to stroke according topreviously published procedures³². Following anesthesia, the carotidartery was accessed using the operative approach previously described indetail³³, including division/coagulation of the occipital andpterygopalatine arteries to obtain improved visualization and vascularaccess. A nylon suture was then introduced into the common carotidartery, and threaded up the internal carotid artery to occlude theorigin of the right middle cerebral artery (MCA). Nylon (polyamide)suture material was obtained from United States Surgical Corporation(Norwalk, Conn.), and consisted of 5.0 nylon/13 mm length for 27-36 gmice, and 6.0 nylon/12 mm length for 22-26 g mice. After 45 minutes ofocclusion, the suture was withdrawn to achieve a reperfused model ofstroke. Although no vessels were tied off after the suture was removed,the external carotid arterial stump was cauterized to prevent frankhemorrhage.

Measurements of relative cerebral blood flow were obtained as previouslyreported³²⁻³⁵ using a straight laser doppler flow probe placed 2 mmposterior to the bregma, and 6 mm to each side of midline using astereotactic micromanipulator, keeping the angle of the probeperpendicular to the cortical surface. These cerebral blood flowmeasurements, expressed as the ratio of ipsilateral to contralateralblood flow, were obtained at baseline, and immediately prior to MCAocclusion, 45 minutes after MCA occlusion, and at several time pointsafter withdrawal of the occluding suture.

Measurement of Cerebral Infarction Volumes: After 24 hours, animals wereeuthanized and their brains rapidly harvested. Infarct volumes weredetermined by staining serial cerebral sections with triphenyltetrazolium chloride and performing computer-based planimetry of thenegatively (infarcted) staining areas to calculate infarct volume (usingNIH image software).

Neurological Exam: Prior to giving anesthesia, mice were examined forneurological deficit 23 h after reperfusion using a four-tiered gradingsystem: a score of 1 was given if the animal demonstrated normalspontaneous movements; a score of 2 was given if the animal was noted tobe turning towards the ipsilateral side; a score of 3 was given if theanimal was observed to spin longitudinally (clockwise when viewed fromthe tail); and, a score of 4 was given if the animal was unresponsive tonoxious stimuli. This scoring system has been previously described inmice³²⁻³⁴, and is based upon similar scoring systems used in rats³⁶.Immunostaining of cerebral cortex following induction of stroke inwild-type mice was performed as described above using a rabbitpolyclonal antibody made using purified recombinant murine ABAD as theimmunogen. Quantitation of microscopic images was accomplished with theUniversal Imaging System.

Results:

Transgenic mice overexpressing RAGE under control of theplatelet-derived growth factor promoter were subjected to transientmiddle cerebral artery occlusion along with non-transgenic littermates.Mice were 26-29 grams in weight and about 10 weeks old. The volume ofinfarcted cerebral tissue was reduced about 50% (P<0.05) in thetransgenic mice compared with the controls (see FIG. 19A shows theresults of studies in all mice, and FIG. 19B shows triphenyl tetrazoliumchloride staining of selected cerebral sections). It is important tonote that glucose levels were monitored in the animals before and afterthe ischemic episode, because of the known effect of hyperlgycemia oninfarct volume. The animals remained normoglycemic throughout theprocedure, These data indicate that overexpression of RAGE in neuronshas a neuroprotective effect. Thus, it would be important to testpotential RAGE inhibitors in such an animal to determine if theyantagonized this protective property of the receptor. Figure legend: forthe figure which you SHOULD send a messenger to pick up. Induction ofstroke in transgenic mice overexpressing RAGE on the platelet-derivedgrowth factor promoter: cerebral infarct volume (FIG. 19A) and resultsof triphenyl tetrazolium chloride staining of selected cerebral sections(FIG. 19B).

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1. A transgenic mouse whose genome comprises a first DNA segmentcomprising: (a) a second DNA segment comprising a platelet derivedgrowth factor B-chain promoter; and (b) a third DNA segment whichencodes a human receptor for advanced glycation endproducts; wherein thesecond DNA segment is operatively linked to the third DNA segment andthe first DNA segment is integrated into the genome of the mouse; andwherein said mouse exhibits a reduced amount of infarcted cerebraltissue following induction of a transient middle cerebral arteryocclusion in the mouse as compared to the amount exhibited by anotherwise identical mouse lacking said first DNA segment.
 2. A methodwhich comprises administering compound to the transgenic mouse of claim1 and determining whether the mouse exhibits an increase in the amountof infarcted cerebral tissue following induction of a transient middlecerebral artery occlusion relative to the amount of infarcted cerebraltissue following such induction in an identical mouse in the absence ofthe compound.